Calcineurin-dependent regulation of renal Na-(K-)Cl-cotransporters (II)

盐和水的体内平衡和血压的调节关键取决于肾小管上皮细胞的功能。远端肾单位（粗升支[TAL]，远曲小管[DCT]）通过盐重吸收有效地决定容量调节。肾脏特异性Na-（K-）Cl-协同转运蛋白（阳离子-氯协同转运蛋白，CCC）包括TAL中的NKCC 2和DCT中的NCC。两者都是由磷酸激酶（WNK/SPAK/OSR 1）级联激活的。级联反应的缺陷会导致体内平衡的改变。在单遗传性假性醛固酮减少症（PHA-II）与管状盐潴留，我们已经确定了WNK 1和WNK 4的功能，建立了一个链接到他们的cullin/kelch样介导的降解，并描述了一个新的，过度激活WNK 1突变体。钙依赖性丝氨酸-苏氨酸磷酸酶钙调神经磷酸酶控制这些激活步骤。钙调磷酸酶抑制剂（CNI;他克莫司、环孢菌素A）在移植后成功地用于免疫抑制，但副作用如容量保留和高血压是常见的。我们已经表明，CNI可能部分表型PHA-II与肾小管盐潴留，这可以通过抑制NCC与噻嗪类利尿剂减少。NKCC 2的参与也被证明。为了分析所涉及的机制，钙调磷酸酶亚型和CNI结合亲免蛋白进行了本地化，和亲免蛋白缺陷的小鼠模型进行了评估。钙调神经磷酸酶，激酶和CCC之间的结合特性的分析，使我们确定了必要的，非冗余的介体蛋白，如膜联蛋白A2的运输和脂筏协会的NKCC 2，以及钙调神经磷酸酶同源蛋白1（CHP 1）和SORLA传递钙调神经磷酸酶对NKCC 2的影响。在更新期间，我们将遵循这样的假设，即钙调神经磷酸酶在TAL和DCT中不同地发挥重要的调节功能。CCC、钙调神经磷酸酶和激酶之间的相互作用将进行机制验证。钙调神经磷酸酶的磷酸酶活性将在分子水平上测定和说明。支架蛋白的作用以及膜联蛋白A2、CHP 1和SORLA的影响将在CCC磷酸化调节的背景下得到验证。蛋白酶体降解，侵略形成和自噬将研究在适应性重建DCT速尿治疗和饮食诱导的运输刺激，考虑到钙调磷酸酶和WNK-1功能的参与。将针对WNK-SPAK/OSR 1-CN-CCC级联的调节适应性分析由高盐低钾饮食（“西方饮食”）引起的容量保持。将研究持续的CNI效应，以登记已识别产品的状态和相互作用，并将考虑内分泌效应。应根据本项目的结果实现解决免疫抑制治疗副作用的治疗方案。

Regulation of salt and water homeostasis and blood pressure critically depends on tubular epithelial functions in the kidney. The distal Nephron (thick ascending limb [TAL], distal convoluted tubule [DCT]) determines volume regulation effectively via salt reabsorption. The kidney-specific Na-(K-)Cl-cotransporters (cation-chloride cotransporters, CCC) comprise NKCC2 in TAL and NCC in DCT. Both are activated by a cascade of phosphokinases (WNK/SPAK/OSR1). Defects in the cascade cause alterations in homeostasis. In monogenetic pseudohypoaldosteronism (PHA-II) with tubular salt retention we have characterized WNK1 and WNK4 functions, established a link to their cullin/kelch-like-mediated degradation, and described a new, overactivating WNK1 mutant. The calcium-dependent serine-theorine phosphatase calcineurin controls these activating steps. Calcineurin Inhibitors (CNI; tacrolimus, cyclosporin A) are successfully administered for immunosuppression after transplantation, but side effects such as volume retention and hypertension are common. We have shown that CNI may in part phenocopy PHA-II with tubular salt retention, which could be reduced by inhibiting NCC with thiazide diuretics. An involvement of NKCC2 was demonstrated as well. To analyze the mechanisms involved, calcineurin isoforms and CNI-binding immunophilins were localized, and an immunophilin-deficient mouse model evaluated. Analysis of the binding properties between calcineurin, the kinases, and the CCC led us to the identification of essential, non-redundant mediator proteins such as annexin A2 for the trafficking and lipid-raft association of NKCC2, as well as calcineurin homologous protein 1 (CHP1) and SORLA to transmit calcineurin effects on NKCC2. During the renewal period, we will follow the hypothesis that calcineurin differntially exerts important regulatory functions in TAL as well as in DCT. The interactions between CCC, calcineurin, and kinases will be validated mechanistically. The phosphatase activity of calcineurin will be determined and specified at the molecular level. The role of scaffold proteins as well as the impact of annexin A2, CHP1 and SORLA will be verified in the context of CCC phosphoregulation. Proteasomal degradation, aggresome formation and autophagy will be studied during adaptive restructuring of DCT upon furosemide therapy and diet-induced transport stimulation, considering an involvement of calcineurin and WNK-1 functions. Volume retention caused by a high-salt, low-potassium diet ("Western diet") will be analyzed with respect to regulatory adaptations of the WNK-SPAK/OSR1-CN-CCC cascade. Sustained CNI effects will be studied to register status and interactions of the identified products, and endocrine effects will be considered. Therapeutic options addressing the side effects of immunosuppressive therapy shall be achieved based on the results of this project.